BENZYL ACETATE
1. EXPLANATION
This substance was evaluated for an acceptable daily intake for
man (ADI) by the joint FAO/WHO Expert Committee on Food Additives at
the eleventh, twenty-seventh, twenty-ninth and thirty-first meetings
(Annex 1, references 14, 62, 70 and 77). A toxicological monograph
was issued in 1968 (Annex 1, reference 15). The ADI of 0-5 mg/kg
bw/day which was established at the eleventh Meeting was made
temporary at the twenty-seventh meeting.
Since the previous evaluation, additional data have become
available and are summarized and discussed in the following monograph.
The previously published monograph has been expanded and is reproduced
in its entirety below.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
This compound is absorbed from the gastrointestinal tract,
through the lungs and through the intact skin. It is hydrolyzed in
man to benzyl alcohol and acetate: the benzyl radical is oxidized to
benzoic acid and excreted as hippuric acid (Snapper et al., 1925).
Benzyl acetate was readily hydrolyzed in vitro with a
pancreatin preparation (Grundschoser, 1977). Benzylmercapturic acid
and hippuric acid were isolated from the urine of rats that had been
injected subcutaneously with benzyl acetate (Clapp & Young, 1970).
Mice and rats were dosed either intravenously or orally with 14C
labelled benzyl acetate. The intravenous dose for mice was equivalent
to 10 mg/kg bw and for rats 5 mg/kg bw. For oral dosing, the benzyl
acetate was dissolved in corn oil and administered at dose levels
equivalent to 10, 100 or 1000 mg/kg bw for mice and 5, 50 and 500
mg/kg bw for rats. Elimination of benzyl acetate as CO2 or volatiles
was minimal. The elimination of the 14C label occurred mainly in the
urine with less than 1% being detected in the feces. More than 90% of
the 14C in the urine was present as hippuric acid, with minor amounts
present as benzyl alcohol and benzylmercapturic acid (up to 4%). No
unchanged benzyl acetate was present in urine. The elimination was
complete within 24 hours. Neither route of administration or dose had
any significant effect on the pattern of elimination. Benzoyl
glucuronide levels were not measured. Repeated dosing of rats with
500 mg/kg bw/day benzyl acetate for 14 days, followed by a single
dose of 14C benzyl acetate did not change the clearance pattern.
Analysis of tissues of animals, sacrificed 24 hours after
administration of 14C either i.v. or orally, failed to detect the
presence of 14C activity (Matthews & Burka, 1984).
Male rats received [methylene-14C]-benzyl acetate by gavage
either in corn oil or in propylene glycol at doses of 5, 250 or 500
mg/kg bw. Radioactivity was measured in feces, urine, plasma and
tissues. Metabolites in urine and plasma were identified by HPLC and
TLC. Absorption of benzyl acetate was more rapid at the lower doses
and in the absence of a vehicle. The bulk of the administered dose
(70-89%) was excreted in the urine over the initial 24-hr period with
very little in the feces (approximately 4%) after 72 hours. The
elimination of benzyl acetate and metabolites was essentially complete
by 3 days, as indicated by negligible residues in tissue, regardless
of whether benzyl acetate was given neat or in corn oil. Since benzyl
acetate was not found in the plasma or urine at any time, the authors
concluded that it was readily hydrolyzed. Small amounts of benzyl
alcohol, the initial product of ester cleavage, was detected only in
plasma samples. At the higher doses, benzoic acid was the major
metabolite in plasma, while at the 5 mg/kg bw dose, hippuric acid (the
glycine conjugate of benzoic acid) predominated. Hippuric acid was
the major metabolite in urine, and the proportion of the original dose
it represented was not significantly affected by dose size. The
proportion of the dose present as benzoyl glucuronide increased with
dosage, leading the authors to conclude that this demonstrated a
limited capacity of the glycine conjugation mechanism (Chidgey &
Caldwell, 1986).
2.2 Toxicological studies
2.2.1 Acute toxicity
Species Sex Route LD50 Reference
(mg/kg bw)
Rat ? oral 2490-3690 Jenner et al., 1967
von Oettingen, 1960
Rabbit ? oral 2640 von Oettingen, 1960
2.2.2 Short-term studies
2.2.2.1 Mice
Groups of 5 mice (B6C3F1) of each sex were administered single
doses of benzyl acetate equivalent to 250, 500, 1000, 2000 or 4000
mg/kg bw in corn oil by gavage. The animals were observed for 15
days. All mice receiving 4000 mg/kg bw and females receiving 2000
mg/kg bw became inactive immediately after dosing. All mice in the
highest dose group, and 1/5 males and 2/5 females dosed at 2000 mg/kg
bw died by day 2 of the study. No other compound-related effects were
reported (NTP, 1986).
Groups of 5 mice (B63F/N) of each sex were dosed with 0, 125,
250, 500, 1000 or 2000 mg/kg bw benzyl acetate in corn oil by gavage
daily for 14 days. On day 16 all surviving animals were killed and
autopsied. All male mice at the highest dose level had died by day 3
of the study. Weight changes were not dose-related. At autopsy the
only effect reported was a roughening of the mucosa of the stomach in
the cardiac region in 2/5 males and all females in the highest dose
group, and 1/5 females in the 1000 mg/kg bw/day group (NTP, 1986).
Groups of 10 mice (B6C3F1) of each sex were administered 0, 125,
250, 500 or 12000 mg/kg bw/day benzyl acetate in corn oil by gavage,
5 times a week for 13 weeks. A total of 7/10 of the females in the
highest dose group died. Compound-related clinical effects were
observed in the highest dose group, included trembling, inactivity,
labored breathing and depressed body temperature. At autopsy, no
gross or microscopic effects were noted (NTP, 1986).
2.2.2.2 Rats
Groups of 15 males and 15 females were fed a mixture of aromatic
esters, including 15.8 mg/kg bw/day of benzyl acetate for 12 weeks.
No adverse effects were noted (Oser, 1967). Groups of 5 rats (F344/N)
approximately 6 weeks old, were administered 0, 250, 500, 1000, 2000
or 4000 mg/kg bw/day benzyl acetate by gavage in corn oil, daily for
14 days. On day 16, surviving animals were killed and autopsied.
None of the rats dosed at 4000 mg/kg bw/day survived beyond 2 days, at
the 2000 mg/kg bw/day level all rats had died within 5 days. No other
deaths were reported. There was a depression of mean body weight gain
relative to controls of more than 10% in the 500 mg/kg bw/day male
group, and in the 1000 mg/kg bw/day group in both males and females.
At autopsy the only effect reported was that the cecum was redder than
normal in 3/5 of the animals in the 4000 mg/kg bw/day group (NTP,
1986).
Groups of 10 rats (F344/N) of each sex were administered 0, 62.5,
125, 250, 500 or 1000 mg/kg bw/day benzyl acetate in corn oil, 5 days
per week for 13 weeks. Male and female rats in the 100 mg/kg bw/day
group and females in the 500 mg/kg bw/day group showed clinical
symptoms including trembling, ataxia and sluggishness. A total of
2/10 males and 1/10 females in the highest dose group had died by day
86. Only male rats in the 1000 mg/kg bw/day group showed a depression
in mean body weight relative to controls (21%). At autopsy a
thickened stomach wall was observed in 2/9 males and 4/10 females in
the high dose group (NTP, 1986).
2.2.3 Long-term/carcinogenicity studies
Lifetime studies in mice and rats with benzyl alcohol are
included in this section since the initial step in metabolism of
benzyl acetate is hydrolysis of the ester to benzyl alcohol (Abdo et
al., 1985; Chidgey et al., 1986; NTP, 1986).
2.2.3.1 Mice
Groups of 50 (B6C3F1) mice of each sex were administered benzyl
acetate in corn oil by gavage at doses equivalent to 500 or 1000 mg/kg
bw/day, 5 days/week for 103 weeks. Vehicle control groups of 50
animals/sex were administered corn oil by gavage. Complete gross
necropsies and histopathological examinations were performed on
animals found dead and on those sacrificed at the end of the study.
Mean body weight gains of dosed and control male mice were
comparable throughout the study. In females, treated mice showed
slightly higher mean body weights after week 20 than those of the
controls. Survival of female mice was markedly reduced in the control
(30%) and low dose groups (36%) in comparison with the high dose group
(60%). The high mortality rate in female mice was associated with
infection, resulting in suppurative inflammation or abscesses of the
ovaries, uterus, mesentery, peritoneum or multiple organs (26/35
control, 14/32 low dose and 8/20 high dose). In males, there were no
significant differences between the survival rate of controls and
either treatment group, although a greater number of control males
died before week 45 than in the treated groups.
Hepatocellular adenomas occurred in male mice in 0/50 control,
5/49 low dose and 13/50 high dose animals, and in female mice in 0/50
control, 0/50 low dose, and 6/50 high dose animals. (The cumulative
historical incidences of hepatocellular adenomas in corn oil gavage
controls from 6 studies from the contract laboratory conducted prior
to 3 August 1984, were 36/298 for male and 11/300 for female B6C3F1
mice). Hepatocellular carcinomas were observed in male mice and in the
high dose female group (Males: control 10/50, low dose 14/49 and high
dose 12/50, and females: 1/50 control, 0/50 low dose, and 4/50 high
dose).
Squamous cell papillomas or carcinomas of the forestomach
occurred in male mice and in the high dose female group. The
incidence in males was 4/49 control, 4/48 low dose, and 11/49 high
dose. For female mice the incidence was 0/50 control, 0/50 low dose
and 4/48 high dose. The historical incidences in corn oil gavage
controls from 6 studies from the contract laboratory were 2/296 for
males and 2/297 for females. Forestomach hyperplasia was also
reported in dosed mice (males: control 1/49, low dose 7/48 and high
dose 22/49, and for females: control 1/50, low dose 6/50, high dose
17/48) (NTP, 1986).
Benzyl alcohol was administered by corn oil gavage to groups of
50 mice (B6C3F1) of either sex, at dosages of 0, 100 or 200 mg/kg
bw/day, 5 days a week for 103 weeks. The neurotoxic effects of benzyl
alcohol (lethargy and staggering) in short-term studies formed the
basis for selection of the doses. Complete gross necropsies and
histopathological examinations were conducted on animals found dead
and on those sacrificed at the end of the study.
Mean body weights for dosed and control mice were comparable
throughout the study. The survival of control females was
significantly lower than those in the high dose group after week 44,
but no other differences in survival were noted between dosed groups
and controls (male: control 68%, low dose 66%, high dose 70%;
female: control 50%, low dose 62%, high dose 72%). No significant
treatment-related effects were noted. There was no association of
treatment with the reported incidences of hepatocellular and
forestomach neoplasias after administration of benzyl acetate (NTP,
1987).
2.2.3.2 Rats
Groups of 50 rats (F344/N) of each sex were administered benzyl
acetate in corn oil by gavage at doses equivalent to 150 or 500 mg/kg
bw/day, 5 days/week for 103 weeks. Vehicle control groups of 50
animals per sex were administered corn oil by gavage. Complete gross
necropsies and histopathological examinations were performed on
animals found dead and on those sacrificed at the end of the study.
Mean body weight gains were comparable between treated and
control groups throughout most of the study. No significant
differences were found in the survival of male and female rats
administered benzyl acetate as compared with controls. In male rats,
76% of the controls, 92% of the low-dose group and 80% of the high-
dose group survived to 104-106 weeks. Survival rates for females at
104-106 weeks were: 80% of the controls, 72% of the low-dose group and
72% of the high-dose group.
The incidence of all malignant epithelial tumours
(cystadenocarcinoma, adenocarcinoma and carcinoma) in the preputial
gland of male rats was elevated in the high dose group (control 1/50,
low dose 1/50, high dose 6/50), but this was without statistical
significance.
The incidence of acinar cell adenomas in the pancreas of male
rats was 22/50 in controls, 27/50 at the low dose, and 37/49 at the
high dose. The incidence at the high dose was significantly greater
than in the vehicle controls (p = 0.001). Acinar cell hyperplasia was
also observed in male rats (37/50 control, 34/50 low dose, and 36/49
high dose). No acinar cell hyperplasia or adenoma of the pancreas was
observed in any of the female animals in the three experimental
groups. There was an increased incidence of retinopathy (not
specified) and cataracts in the high dose males and low dose females.
However, this effect was attributed by the authors to the proximity of
these rats to fluorescent light (NTP, 1986).
Benzyl alcohol was administered by corn oil gavage to groups of
50 F344/N rats of each sex at dosages of 0, 200 or 400 mg/kg bw/day,
5 days a week for 103 weeks. Complete gross necropsies and
histopathological examinations were conducted on animals found dead
and on those sacrificed at the end of the study.
Mean body weights of dosed and vehicle control male and female
rats were comparable throughout the study. No compound-related
clinical signs were observed, although at month 3 a
sialodacryoadenitis virus infection was widespread among the study
animals. The survival of animals in both dosed female groups was
significantly lower than that of vehicle controls (70% - control; 34%
- low dose; 34% - high dose), which was the result of a much higher
incidence of accidental gavage-related deaths in these groups.
Survival among male rats was comparable in all groups (control - 56%;
low dose - 54%; high dose - 48%).
Cataracts and retinal atrophy were observed at increased
incidences in the high dose rats in this study. This was again
attributed by the authors to the proximity of this group of animals to
fluorescent light for most of the study. Hyperplasia of the
forestomach epithelium was noted at an increased incidence in male
rats (control 0/48, low dose 0/19, high dose 4/50). Hemorrhage and
foreign material in the respiratory tract were noted in dosed rats
which died prior to the terminal kill. The authors suggested this
could have been the result either of direct deposition of material
into the lung due to gavage "accidents" or of the anesthetic
properties of benzyl alcohol resulting in reflux of gavage material,
and aspiration into the lungs. No pancreatic acinar cell adenomas
were reported in any of the test or control groups in contrast to the
results from the corn oil gavage study in rats with benzyl acetate.
No other effects of treatment were noted (NTP, 1987).
2.2.5 Special studies on genotoxicity
Results of genotoxicity assays on benzyl acetate
Test System Test Object Concentration Results Reference
of benzyl acetate
Ames test (1) S.typhimurium 0-10,000 µg/ Negative NTP,1986;
TA100,TA1535, plate Mortelmans
TA1537, TA98 et al., 1986
In vitro Chinese 50-5,000 µg/ml Negative NTP, 1986
mammalian cytogenicity hamster
(SCE, chromosomal ovary cells
aberrations) (2)
In vitro Mouse lymphoma 0.25-1.25 µg/ml Positive NTP, 1986
mammalian gene cells (3)
mutation (2) (L5178Y/Tk±)
In vitro Bacillus 21 µg/disk Negative Oda et al.,
bacterial subtilis 1978
gene mutation
Unscheduled DNA Rat hepatocytes Negative Mirsalis et al.,
synthesis 1983
in vivo and
in vitro)
(1) in the presence or absence of induced rat or hamster liver S-9 fractions
(2) in the presence or absence of induced rat liver S-9 fraction
(3) positive in the presence of metabolic activation, negative in the absence of metabolic
activation.
3. COMMENTS
At the thirty-first meeting the Committee extended the temporary
ADI of 0-5 mg per kg bw pending the evaluation of lifetime gavage
studies with benzyl alcohol, a normal metabolite of benzyl acetate.
These studies did not reveal the increased incidence of either
hepatocellular or forestomach tumours in mice or pancreatic tumours in
rats which had previously been observed in studies with benzyl
acetate.
There are difficulties in interpreting the carcinogenicity
studies with benzyl acetate that were conducted by gavage. Since it
is known that new long-term studies are underway with benzyl acetate
incorporated into the diet of rats and mice, the present Committee
decided to extend the temporary ADI of 0-5 mg/kg bw until 1993 pending
the evaluation of the results of the new studies.
In view of one positive report of mutagenic activity in vitro,
it would be desirable to ascertain whether results of an in vivo
study demonstrating the lack of the induction of unscheduled DNA
synthesis can be confirmed by an in vivo test for chromosome damage
in bone marrow.
4. EVALUATION
Level causing no toxicological effect
Rat: 15.8 mg/kg bw/day.
Estimate of temporary acceptable daily intake
0-5 mg/kg bw.
Further work or information
Required (by 1993):
Oncogenicity studies in rats and mice.
Desired:
In vivo study for chromosome damage in bone marrow.
5. REFERENCES
ABDO, K.M. HUFF, J.E., HASEMAN, J.K., BOORMAN, G.A., EUSTIS, S.L.,
MATTHEWS, H.B., BURKA, L.T., PREJEAN, J.D. & THOMPSON, R.B. (1985).
Benzyl acetate carcinogenicity, metabolism and disposition in Fischer
344 rats and B6C3F1 mice. Toxicology, 37, 159-170.
CHIDGEY, M.A.J. & CALDWELL, J. (1986). Studies on benzyl acetate. I.
Effect of dose size and vehicle on the plasma pharmacokinetics and
metabolism of [methylene-14] benzyl acetate in the rat. Food
Chem.Toxic., 2, 1257-1265.
CHIDGEY, M.A.J., KENNEDY, J.F. & CALDWELL, J. (1986). Studies on
benzyl acetate. II. Use of specific metabolic inhibitors to define the
pathway leading to the formation of benzylmercapturic acid in the rat.
Food Chem.Toxicol., 24, 1267-1271.
CLAPP, J.J. & YOUNG, L. (1970). Formation of mercaptic acid in rats
after administration of aralkyl esters. Biochem.J., 118, 765-771.
GRUNDSCHOSER, F. (1977). Toxicological assessment of flavouring
esters. Toxicology, 8, 387-390.
JENNER, P., HAGAN, E., TAYLOR, J., COOK, E. & FITZHUGH, O.G. (1967).
Food flavourings and compounds of related structure, I. Acute Oral
Toxicity. Food Cosmet.Toxicol., 2, 327-343.
MATTHEWS, H.B. & BURKA, L.T. (1984). Benzyl acetate metabolism and
disposition in rats and mice, NTP (1984). NIH Publication No. 82-2506,
USDHSS.
MIRSALIS, J., TYSON, K., BECK, J., LOH, F., STEINMETZ, K., CONTRERAS,
C., AUSTERE, L., MARTIN, S. & SPALDING, J. (1983). Induction of
unscheduled DNA synthesis (UDS) in hepatocytes following in vitro
and in vivo treatment (Abstract No. Ef-5). Environ.Mutagenesis, 5,
482.
MORTELMANS, K., HAWORTH, S., LAWLOR, T., SPECK, W., TAINER, B. &
ZEIGER, E. (1986). Salmonella mutagenicity tests: II. Results from
the testing of 270 chemicals. Environmental Mutagenesis, 8, 1-119.
NTP (1986). NTP technical report on the toxicology and carcinogenesis
studies of benzyl acetate (CAS NO. 140-11-4) in F344/N rats and B6C3F1
mice (gavage studies). NIH Publication No. 86-2506, U.S. Department of
Health and Human Services, Public Health Service, National Institutes
of Health, Research Triangle Park, NC.
NTP (1987). Board Draft. NTP technical report on the toxicology and
carcinogenesis studies of benzyl alcohol (CAS NO. 100-51-6) in F344/N
(gavage studies). NIH Publication No. 88-2599, U.S. Department of
Health and Human Services, Public Health Service, National Institutes
of Health, Research Triangle Park, NC.
ODA, Y., HAMANO, Y., INQUE, K., YAMAMOTO, H., NIIHARA, T. & KUNITA, N.
(1978). Mutagenicity of food flavours in bacteria. Osaka-furitsu
Kosnu Eisei Kenkyu Hokoku snokunin eisein nem, 9, 177-181.
OSER, B.L. (1967). Unpublished report.
SNAPPER, I., GRUNBAUM, A. & STRUKOP, S. (1925). Biochem.Z., 155,
163.
VON OETTINGEN, W.W. (1960). AMA Arch.Ind. Health, 21, 28.